3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Detection and molecular epidemiology of ciprofloxacin-resistant Neisseria gonorrhoeae, using a real-time polymerase chain reaction (PCR)(2011-03-22) Magooa, Mahlape PreciousEmergence and spread of resistance to ciprofloxacin among Neisseria gonorrhoeae strains has reduced the options of effective treatment for gonococcal infections and has become a concern worldwide. Up until 2008, ciprofloxacin was recommended first-line therapy for treatment of presumptive N. gonorrhoeae infections in South Africa. At the time this MSc project was conceived, ciprofloxacin was still used as first-line therapy for presumptive gonococcal infections. A real-time polymerase chain reaction (PCR) assay was used to detect ciprofloxacin-resistant N. gonorrhoeae in DNA extracted from non-invasive urine samples collected as part of the national microbiological surveillance (NMS) programme during 2006-2007. The molecular epidemiology of ciprofloxacinresistant Neisseria gonorrhoeae was investigated by sequencing the quinolone resistance determining regions (QRDR) of the gyrA and parC genes of N. gonorrhoeae and performing N. gonorrhoeae multi-antigen sequence typing (NGMAST). As part of the NMS program for sexually transmitted infections (STIs) urine and urethral swabs were collected from men presenting with urethral discharge at primary health care clinics in Johannesburg (Gauteng), Cape Town (Western Cape) and Kimberley (Northern Cape). Urine samples and cultured N. gonorrhoeae isolates from 2006-2007 were stored at -700C and available for this study. Gonococci, previously isolated from urethral swabs, were subcultured directly onto New York City media. Isolate identity was re-confirmed by typical colony morphology and biochemical tests. Urine samples from Johannesburg were tested in order to develop the real-time PCR protocol. Subsequently, paired urethral swab DNA and N. gonorrhoeae cultures were tested from NMS patients recruited in Kimberley and Cape Town. Where possible, the PCR assay results were compared with paired antibiotic susceptibility data for ciprofloxacin. Quinolone resistance determining regions (QRDR) for gyrA and parC were screened for known point mutations associated with resistance to ciprofloxacin. Detection of mutations by the real-time PCR assay generally agreed with the phenotype of either decreased susceptibility or resistance to ciprofloxacin. All ciprofloxacin resistant gonococcal isolates had the same gyrA and parC mutations, which initially suggested that quinolone resistant N. gonorrhoeae (QRNG) in Kimberley, Cape Town and Johannesburg, may be attributed to the spread of a single clone. The use of a more discriminatory typing scheme, Neisseria gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST) genotyping, revealed that ciprofloxacin resistant gonococcal isolates in Johannesburg and Cape Town were heterogeneous, with sequence type (ST) 217 being most prevalent in both cities (5/16, Johannesburg; 7/11, Cape Town). In contrast, all eight QRNG isolates from Kimberley were typed as ST 533. The use of molecular methods allowed ciprofloxacin antimicrobial susceptibility determination by PCR in non-invasive specimens. This is useful in situations where bacterial cultures are unavailable or die before antimicrobial susceptibility testing can be performed. Molecular assays to detect ciprofloxacin resistance may guide physicians as to the most ideal antimicrobial combinations for individual patient treatment. As a result of emerging widespread resistance gonococci to ciprofloxacin, in 2008, the Department of Health recommended that ciprofloxacin be removed as a first line therapy in the South African national sexually transmitted infections treatment guidelines for treatment of urethritis, cervicitis and their complications. Although ciprofloxacin is no longer used as a first-line therapy to treat gonorrhoea within our country, it may still be used in cases of severe penicillin allergy or as part of multi-drug therapy for gonococcal infections in the future. The ability to detect ciprofloxacin resistance by real-time PCR will be a useful technique in such situations.Item Development of a real-time PCR incorporating high resolution melting analysis to screen HIV-1 samples for resistance-related codons(2011-02-01) Sacks, DavidIntroduction High resolution melting analysis (HRMA) accurately, rapidly and cost effectively detects single nucleotide polymorphisms by monitoring DNA dissociation kinetics. This technology was applied to HIV samples to assess whether it could be used to detect clinically relevant drug resistance mutations. Methods HRMA-PCR assays incorporating unlabeled probes were designed to genotype 12 mutation codons in the HIV-1 p66/p51 of engineered plasmids and 116 HIV-1 samples. Results HRMA correctly genotyped 63%-88% of the K103N, Y181C, M184V, Q151M and G190A mutations. Each assay had a 1.7%-3.4% discordance, most of which was due to the increased analytical sensitivity of HRMA (~5-20%). Only mutant K65R and V106M were correctly identified while the 41, 67, 70, 215 and 225 codons could not be genotyped. Assay modifications had some success in masking the affects of polymorphisms. Conclusion These assays can be used for genotyping selected major HIV-1 resistance mutations and should be further developed as a resistance surveillance tool.Item Antimicrobial susceptibility of anaerobic organisms isolated from clinical specimens at Charlotte Maxeke Johannesburg Academic Hospital(2010-04-15T11:22:30Z) Naidoo, SudeshniAnaerobic bacteria cause serious life-threatening infections such as endocarditis, sepsis, intra abdominal, pleuro-pulmonary and central nervous systems infections. Most infections are polymicrobial and involve aerobes and anaerobes. Empiric therapy is generally based on the expected pathogens and the particular type of infection. Even when specimens are cultured and anaerobes identified, not all laboratories perform susceptibility testing. The clinician often relies on published surveillance data when selecting treatment regimens. Antimicrobial susceptibility of anaerobic bacteria is becoming increasingly unpredictable. Resistance can vary significantly and patterns differ geographically, and even within units of the same hospital. From June 2005 until February 2007, 180 consecutive anaerobes isolated from relevant, non- repetitive clinical specimens were tested routinely with the E test method for susceptibility to amoxicillin/ clavulanate (XL), clindamycin (Cm), metronidazole (Mz), penicillin (Pg), ertapenem (Etp), cefoxitin (Fx), ceftriaxone (Tx), chloramphenicol (Cl), and piperacillin/tazobactam (Ptc). The results were read after 48hr incubation in anaerobic conditions. Specimen distribution was as follows: abdominal fluid (3), abscess (7), abdominal abscess (4), aspirates (3), blood cultures (27), bone (3), breast (3), drainage fluid (2), empyema (1), fluids (36), other (4), placental (1), pleural fluid (2), pus (41), tissues (34), umbilicus (1) and unknown sites (8). Bacteroides fragilis was isolated from 81 (45%) clinically significant specimens, followed by Clostridium perfringens 23 (13%), Peptostreptococcus anaerobius 15 (8%) and Prevotella melaniniogenicus 15 (8%). B. fragilis demonstrated a 97.5% resistance to penicillin and 12.3% resistance to metronidazole. C. perfringens exhibited no resistance to penicillin and metronidazole while P. anaerobius had 40% resistance to penicillin and no resistance to metronidazole. P. melaninogenicus was resistant to penicillin in 60% and 6.7% to metronidazole. Overall, chloramphenicol, piperacillin/tazobactam, ertapenem and amoxicillin/clavulanate demonstrated the highest activity to anaerobic isolates, 100%, 99.4%, 97.2% and 96.7%, respectively. Among the 180 tested anaerobes a total of 8.9% resistance has been observed with metronidazole and 81.7% sensitivity with clindamycin. Periodic surveillance to monitor the susceptibility profile of the B. fragilis group and other anaerobic organisms is recommended to create empirical guidelines for appropriate use of antimicrobial agents.Item The role of Aktin the prevention of Apoptosis in HL-60 cells, A human leukaemic cell line(2006-10-25T13:48:51Z) Drummond, Chantal, PaulaStudies on the development of drug resistance in several cancer types, including acute myeloid leukaemia (AML), have implicated the PI3-kinase pathway. This pathway phosphorylates Akt resulting in the activation of proteins involved in cell survival. The aim of this study is to determine the role that Akt plays in urvival and the relationship between Akt, IKK and IkB in HL-60 cells. This study demonstrated that etoposide caused apoptosis in HL-60 cells, which was slightly increased when the PI3-kinase pathway was inhibited by LY294002. Stimulation with PDGF resulted in cell proliferation and increased Akt, IKK and IkB phosphorylation. Although pre-treatment with LY294002 decreased the amount of Phospho-Akt, phosphorylation of IKK and IkB still occurred. Therefore additional pathways must be involved in IkB regulation in HL-60 cells. Akt mRNA transcription was decreased when the cells were pretreated with LY294002 and either PDGF or etoposide. In conclusion, the PI3-kinase pathway plays a minor role in the survival of HL-60 cells and Akt substrates other than IKK are mediating this survival.